Coagulation and fibrinolysis are physiological pathways which are involved in maintaining normal blood hemostasis in mammals. Under conditions in which a vascular injury occurs, the coagulation pathway is stimulated to form a blood clot to prevent the loss of blood. Immediately after the vascular injury occurs, blood platelets begin to aggregate at the site of injury forming a physical plug to stop the leakage. Additionally, the injured vessel undergoes vasoconstriction to reduce the blood flow to the area and fibrin begins to aggregate forming an insoluble network or clot which covers the ruptured area. Once the vessel has been repaired, the fibrinolysis pathway is stimulated to dissolve the clot.
The balance between the coagulation and the fibrinolytic pathways is essential to the protection of the mammal from excessive blood loss and excessive fibrin build-up in the vascular system which would prevent normal blood flow. When an imbalance is created between the coagulation and fibrinolytic pathways, the physiological result in the mammal is often the development of a serious disease state. When the imbalance in the pathways shifts towards excessive coagulation, the result is the development of thrombotic tendencies which are often manifested as heart attacks, strokes, deep vein thrombosis and myocardial infarcts. When the balance shifts towards excessive fibrinolysis and decreased coagulation, the result is hemorrhage.
The coagulation pathway comprises a series of enzymatic activations which result in the formation of a fibrin clot. The tetrameric complex known as prothrombinase is an essential enzyme in the coagulation pathway. Prothrombinase is composed of a negatively-charged surface, Ca.sup.2+, factor Xa (the enzyme) and factor Va (essential cofactor). Loss or inactivation of factor Va or factor Xa results in a loss in prothrombinase activity. The active prothrombinase enzyme catalyzes the conversion of prothrombin to thrombin. Thrombin catalyzes the cleavage of fibrinogen releasing fibrinopeptides A and B resulting in the production of fibrin monomer which is capable of spontaneously polymerizing to form a clot.
The fibrinolytic pathway also comprises a series of enzymatic activations but which terminate in the solubilization of a fibrin clot resulting in the removal of fibrin clots after vascular injury repair. Plasminogen, which is the zymogen, or inactive, plasma form of plasmin, has an affinity for fibrin clots and forms complexes with fibrin throughout various regions of the porous fibrin network. Another protease, tissue plasminogen activator (t-PA) cleaves a specific bond within the plasminogen zymogen resulting in the formation of the active form of the enzyme, plasmin. t-PA, however, is a poor activator of plasminogen in the absence of fibrin. T-PA, in combination with the fibrin, is a potent activator of plasminogen. The resulting plasmin partially degrades fibrin exposing C-terminal lysines on the fibrin. This partially degraded fibrin is a particularly potent cofactor for the t-PA-induced activation of plasminogen. Further degradation leads to the solubilization and degradation of the fibrin clot.
Although thrombin is an essential component of the anticoagulation pathway (directly causing the cleavage of fibrinogen to fibrin), in the presence of a specific cofactor, thrombomodulin, thrombin assumes another role which leads to the downregulation of thrombin and thus the downregulation of the coagulation pathway. In the presence of thrombomodulin, which is a component of the blood vessel wall, thrombin develops a specificity for protein C and catalyzes the proteolytic activation of protein C to form serine protease activated protein C (APC). APC proteolytically inactivates the essential cofactors, factor Va and factor Villa resulting in the downregulation of the coagulation pathway. By inactivating cofactor Va, APC causes a loss in prothrombinase activity. Because active prothrombinase catalyzes the production of thrombin from prothrombin, the APC-induced loss in prothrombinase activity results in a decreased production of thrombin. The net result of this pathway is the negative feedback regulation of thrombin when thrombomodulin binds to thrombin.
Thrombin, thrombomodulin, and APC are critical factors in the balance between the coagulation and fibrinolytic pathways because in addition to being important components of the coagulation pathway, these components are also involved in the fibrinolytic pathway. Either high levels of thrombin and/or when thrombin is associated with the cofactor thrombomodulin, thrombin activates a fibrinolysis inhibitor, known as thrombin-activatable fibrinolysis inhibitor (TAFI, also known as procarboxypeptidase B, carboxypeptidase B, plasma carboxypeptidase B, carboxypeptidase U, or carboxypeptidase R). TAFI is ordinarily present in the plasma in a zymogen, or inactive, form. Thrombin/thrombomodulin catalyze the activation of TAFI to produce an active TAFI which has been demonstrated to inhibit fibrinolysis. Active TAFI inhibits plasminogen activation by removing C-terminal lysines from partially degraded fibrin. The fibrin which has been cleaved by active TAFI is less effective as a cofactor for a plasminogen activator, such as t-PA dependent activation of plasminogen, than non-cleaved fibrin. Because the potency of fibrin which is available to act as a cofactor is reduced, the amount of plasminogen activated to form plasmin is also reduced, thereby attenuating the solubilization of fibrin and the dissolution of fibrin clots. APC, which is also produced by thrombin/thrombomodulin, inhibits the activation of TAFI by inhibiting the production of thrombin (i.e., by the negative feedback loop-involving loss of prothrombinase activity). Therefore, thrombin, in the absence of thrombomodulin, is involved in the coagulation pathway and in the presence of thrombomodulin, thrombin is involved in the downregulation of the coagulation pathway and in promoting a balance between opposing factors which are involved in the regulation of fibrinolysis (active TAFI inhibits fibrinolysis and APC promotes fibrinolysis by inhibiting the activation of TAFI).
Current therapies for treating disorders associated with imbalances in the coagulation and fibrinolytic pathways involve many risks and must be carefully controlled so that the balance between the pathways is not shifted too far in the other direction. For example, thrombolytic agents such as plasminogen, streptokinase, staphylokinase, t-PA and urokinase are all agents which promote fibrinolysis when there is a risk of thrombosis, but each therapy is also associated with hemorrhagic toxicity. Hemorrhage due to thrombolytic agents results from two factors: (1) the lysis of fibrin at sites of vascular injury prior to the repair of the injury site which may cause bleeding; and (2) a systemic lytic state that results from the systemic formation of plasmin and destruction of other coagulation factors. Therefore, the administration of thrombolytic agents must be carefully monitored in order to provide enough therapeutic agent to result in the destruction of unwanted blood clots, but to prevent the administration of too much therapeutic agent which would cause hemorrhage. In cases of excessive bleeding when coagulants are administered to therapeutically aid in the repair of damaged tissue, there is a risk of forming blood clots due to an overactivation of the coagulation pathway.